This proposed application for a K08 Mentored Clinical Scientist Research Career Development Award is being submitted by Dr. Orapin Horst, a dentist-scientist at the University of California, San Francisco. This award will provide the support and training necessary for Dr. Horst to receive the mentoring to develop into an independent investigator, whereupon she will pursue her long-term goal of creating cell-based therapies and tissue engineering approaches to regenerate the dentin-pulp complex in a manner recapitulating normal development. She will have access to the many opportunities of UCSF to help her accomplish the following goals related to her career development: 1) to learn the disciplines of developmental biology and G protein coupled receptor (GPCR) signaling;2) to experience interdisciplinary research with mentors experienced in basic to clinical translational work;3) to build academic skills to become an outstanding research mentor and faculty member;4) to develop skills in grantsmanship and obtaining funding;and 5) to collect pilot data for a future R01 application. To continue her progress towards these goals, Dr. Horst proposes a research plan that will elucidate signaling events between dental epithelial and mesenchymal progenitor cells to induce odontoblast differentiation and control the formation of the dentin-pulp complex. She will study these signaling events through the function of GPCRs, which have the potential to elicit or modify essentially any signaling pathway. Related findings have been reported in other cell and tissue types, in particular bone, but very little is known about GPCR function in teeth. The central hypothesis that will be tested in this K08 is that GPCR-modulated signaling molecules regulate odontoblast differentiation and dentin matrix formation. This hypothesis is addressed by the following specific aims: 1) To determine how Gs-coupled GPCR signals in dental mesenchymal cells including odontoblasts and preodontoblasts control dentin formation, and 2) To determine how Gs-coupled GPCR signals from dental epithelial cells modulate odontoblast differentiation. These aims will be pursued using a newly developed transgenic mouse model of G-protein signaling in mineralized tissue formation. Transgenic mice with increased Gs-coupled GPCR signals in odontoblasts and preodontoblasts driven by the type I collagen promoter will be used in Specific Aim 1, while mice with increased Gs-coupled GPCR signals in dental epithelial cells driven by the keratin 5 promoter will be used in Specific Aim 2. These studies will identify new interactions between GPCR signaling pathways and other mechanisms regulating odontoblast development and dentin matrix formation. Ultimately, these data will lay a foundation for an interdisciplinary research program and a R01 grant application to engineer a physiologic complex of tubular dentin supporting odontoblasts and pulp to heal carious teeth. The knowledge from this work will also improve our conceptual understanding of genetic dental malformations and environmental factors required for proper cell-matrix interactions to support normal development of dentin and other mineralized tissues. The proposed career development and research activities will take place in highly supportive, research- intensive environments. Two international experts in the developmental biology of teeth, Pamela DenBesten DDS, MS (primary mentor) and Ophir Klein MD, PhD (co-mentor), will supervise Dr. Horst's research focus on epithelial-mesenchymal interactions and formation of mineralized tissues. Two international experts in GPCR signaling, Bruce Conklin MD (co-mentor, GPCR signaling in stem cells) and Robert Nissenson PhD (co- mentor, GPCR signaling in bone), will supervise work with the transgenic mouse models, and relate their vast experience with other stem cell types and mineralized tissues, to understand the role of GPCR signaling in dental stem cells and the formation of the dentin-pulp complex. The mentors are all members of the new Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research of UCSF, which will serve as a central resource for training opportunities (workshops, seminars, journal clubs, and retreats) in combining expertise in developmental, molecular, and cellular biology with medically relevant foci such as that proposed in this project. The proposed research is part of a coordinated career development plan to prepare the candidate to be an outstanding, independent dentist-scientist through research, coursework, and tutorials. The interdisciplinary mentorship team will mentor the career development activities together, and facilitate Dr. Horst's natural progression from current research experiences to the mentored clinical scientist development phase and then to independence. PUBLIC HEALTH RELEVANCE: This K08 application will provide an optimal mentored environment and research training to direct Dr. Horst's further development as an independent clinician scientist. The proposed research plan is to determine the role of G protein signaling in guiding the development of tooth progenitor cells and the formation of the dentin-pulp complex. Completion of this project will lay the groundwork for novel strategies to guide the regeneration and repair of dentin.